title: Novel high-throughput screen against Candida albicans identifies antifungal potentiators and agents effective against biofilms.
abstract: OBJECTIVE
  Microbial adhesion and biofilms have important implications for human health and disease. Candida albicans is an opportunistic pathogen which forms drug-resistant biofilms that contribute to the recalcitrance of disease. We have developed a high-throughput screen for potentiators of clotrimazole, a common therapy for Candida infections, including vaginitis and thrush. The screen was performed against C. albicans biofilms grown in microtitre plates in order to target the most resilient forms of the pathogen.
  
  
  METHODS
  Biofilm growth, in individual wells of 384-well plates, was measured using the metabolic indicator alamarBlue® and found to be very consistent and reproducible. This assay was used to test the effect of more than 120 000 small molecule compounds from the NIH Molecular Libraries Small Molecule Repository, and compounds that enhanced the activity of clotrimazole or acted on the biofilms alone were identified as hits.
  
  
  RESULTS
  Nineteen compounds (0.016% hit rate) were identified and found to cause more than 30% metabolic inhibition of biofilms compared with clotrimazole alone, which had a modest effect on biofilm viability at the concentration tested. Hits were confirmed for activity against biofilms with dose-response measurements. Several compounds had increased activity in combination with clotrimazole, including a 1,3-benzothiazole scaffold that exhibited a >100-fold improvement against biofilms of three separate C. albicans isolates. Cytotoxicity experiments using human fibroblasts confirmed the presence of lead molecules with favourable antifungal activity relative to cytotoxicity.
  
  
  CONCLUSIONS
  We have validated a novel approach to identify antifungal potentiators and completed a high-throughput screen to identify small molecules with activity against C. albicans biofilms. These small molecules may specifically target the biofilm and make currently available antifungals more effective.
authors.affiliation: Antimicrobial Discovery Center and Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02118, USA. m.lafleur@arietiscorp.com
authors.forename: Michael D
authors.initials: MD
authors.lastname: LaFleur
authors.forename: Edinson
authors.initials: E
authors.lastname: Lucumi
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Napper
authors.forename: Scott L
authors.initials: SL
authors.lastname: Diamond
authors.forename: Kim
authors.initials: K
authors.lastname: Lewis
mesh_terms: D000935:Antifungal Agents / Q000302:isolation & purification* / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects*; D002176:Candida albicans / Q000187:drug effects*; D002448:Cell Adhesion / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003022:Clotrimazole / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004347:Drug Interactions; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008826:Microbial Sensitivity Tests; D010078:Oxazines / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; D003022:Clotrimazole